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Tulsi: An important medicinal plant

1.1 General introduction:

History of many drugs can be traced back to their natural origin. At the very dawn of civilization, coexistence of disease with the emergence of life in the earth compelled the primitive man to search a cure from his surroundings. Since then plant was being used as remedy for many diverse disease ranging from simple skin infection to such formidable foes as heart diseases and cancers. The medicinal use of some plants by Indo-Aryans was noted in the Rig Veda (4500-1600BC) and the medicinal value of many plant constituents used by Egyptians was recorded in Papyrus Abus (1500BC).The great physician Hippocrates, who is called ‘the father of medicinal science’, also used plants for treatment of many diseases. In this respect medicine can be considered as an ancient art consisting of plant materials.1

Starting from the stone-age, the use of plants as traditional medicine has increased with the age of civilization. Herbal medicines are still in use in many societies. Although most of the modern medicines are simple compounds, but in many cases the drugs have been originated from the nature, more specifically from plant sources. Plants are considered as natural chemical factory, because synthetic process in biological systems particularly in plants is going on by nature’s ordinary condition of temperature and pressure. The laboratory synthesis of anti-malarial drug quinine requires an intensive work extending over half a century but chicona plant can do it everyday without difficulty. Till today, extensive phytochemical analysis of plants yielded diversified chemical compounds as steroids, terpenoids, flavonoids, chalcones, alkaloids, glycosides etc. clinically used plant metabolites such as taxol isolated from Taxes brevifolia, Vineristine and Vinblastine obtained from Vinca rosea Linn. and digitalis derived from Digitalis purpura are only few of the many striking examples of developing life saving drugs from plant sources.2

Once the plant medication was provided to the ancient people in the crude form, it often exhibited many unwanted effects due to the presence of some toxic components beyond the active constituent. Extensive phytochemical investigation and isolation of active component in the pure form thus became necessary to avoid untoward effects and to ensure safe use of herbal medicines, phytochemical studies of medicinal plants got a rapid pace and the presence of many chemical compounds of diversified nature including many new compounds came in light. These plant derived compounds often played an important role in directing laboratory synthesis of many new classes of drug molecules. In some cases, the plant components became the starting material in the synthetic process of industrial production of many drug molecules. For example, the use of sterol diosgenin, isolated from Mexican yam, for laboratory synthesis of oral contraceptive progesterone reduce the cost of progesterone from a value of $80 per gram to $1.75 per gram.3

Sometimes phytochemical analysis for many plants yielded such chemical constituent having no remarkable therapeutic interest. Sometimes the crude drugs containing several constituents were found to be ineffective in case of therapy for which it is used. The phytochemical investigation of periwinkle plant (Vinca rosea), once used traditionally as an anti-diabetic drug, was found to contain alkaloidal constituents having hypoglycemic potency in minute quantities. The dried seeds of plant Amni visanaga was used as diuretic and antispasmodic in renal collie in Eastern Mediterranean countries and in Arabia, but the research carried out by G.V. Anrep and coworkers resulted in the isolation of Khelin, a component having

vasodilator effect. Khelin appeared in an anti-anginel drug after subsequent clinical trial. The research on Ranwolfia serpentine, which was traditionally used as an antidote for snake bite, revealed the presence of an antihypertensive agent reserpine. Thus systemic research with medicinal plant might open the door of many unknown therapeutic choices. Serendipity has also much impact in discovery of drug leads from natural origin.4

From the above discussion it is evident that the search of plant constituents having therapeutic interest requires bioactivity studies with the crude extracts prior to phytochemical investigation. Only the bioactive extracts or fraction would be of interest for next phytochemical analysis. Without prior biological studies, the phytochemical analysis alone can provide the chemical composition of the plant which may or may not have therapeutic interest. Thus in conducting research on medicinal plants, bioactivity guided phytochemical analysis might be a rational approach.5

1.2 Plant Based Drugs and Medicines:

Today there are at least 120 distinct chemical substances derived from plants that are considered as important active compounds for preparation of drugs currently in use in one or more countries in the world. These chemical substances are shown in the table below. Several of the drugs sold today are simple synthetic modifications or copies of the naturally obtained substances. The original plant substance/chemical name is shown under the "Drug" column rather than the finished patented drug name. For example, many years ago a plant chemical was discovered in a tropical plant, Cephaelis ipecacuanha, and the chemical was named emetine.6 a drug was developed from this plant chemical called Ipecac which was used for many years to induce vomiting mostly if someone accidentally swallowed a poisonous or harmful substance. Ipecac can still be found in pharmacies in many third world countries but has been mostly replaced by other drugs in the United States. Another example of this is the plant chemical named taxol shown in the drug column below. The name taxol is the name of the plant chemical originally discovered in the plant. A pharmaceutical company copied this chemical and patented a drug named Paclitaxe which is used in various types of tumors today in the U.S. and many other countries.7-8

The 120 substances given in table-1 are sold as drugs worldwide but not in all countries. Some European countries regulate herbal substances and products differently than in the United States. Many European countries, including Germany, regulate herbal products as drugs and pharmaceutical companies prepare plant based drugs simply by extracting out the active chemicals from the plants.9

Some of the drug/chemicals shown in table-1 are still sold as plant based drugs requiring the processing of the actual plant material. Others have been chemically copied or synthesized by laboratories and no plant materials are used in the manufacture of the drug. A good example of this is the plant chemical quinine, which was discovered in a rainforest tree (Cinchona ledgeriana) over 100 years ago. For many years the quinine chemical was extracted from the bark of this tree and processed into pills to treat malaria. Then a scientist was able to synthesize or copy this plant alkaloid into a chemical drug without using the original tree bark for manufacturing the drug. Today, all quinine drugs sold are manufactured chemically without the use of any tree bark. However, another chemical in the tree called quinidine which was found to be useful for various heart conditions couldn't be completely copied in the laboratory and the tree bark is still harvested and used to extract this plant chemical from it. Quinidine extracted from the bark is still used today to produce quinidine-based drugs.10

Table-1: List of drugs or chemicals of plant origin having clinical use.

Drug/Chemical Action/Clinical Use Plant SourceAcetyldigoxin Cardiotonic Digitalis lanataAdoniside Cardiotonic Adonis vernalisAescin Anti-inflammatory Aesculus hippocastanumAesculetin Anti-dysentery Frazinus rhychophyllaAgrimophol Anthelmintic Agrimonia supatoriaAjmalicine Circulatory Disorders Rauvolfia sepentinaAllantoin Vulnerary Several plantsAllyl isothiocyanate Rubefacient Brassica nigraAnabesine Skeletal muscle relaxant Anabasis sphyllaAndrographolide Baccillary dysentery Andrographis paniculataAnisodamine Anticholinergic Anisodus tanguticusAnisodine Anticholinergic Anisodus tanguticusArecoline Anthelmintic Areca catechuAsiaticoside Vulnerary Centella asiaticaAtropine Anticholinergic Atropa belladonnaBenzyl benzoate Scabicide Several plantsBerberine Bacillary dysentery Berberis vulgarisBergenin Antitussive Ardisia japonicaBetulinic acid Anticancerous Betula albaBorneol Antipyretic, analgesic, anti-

inflammatorySeveral plants

Bromelain Anti-inflammatory, proteolytic

Ananas comosus

Caffeine CNS stimulant Camellia sinensisCamphor Rubefacient Cinnamomum camphoraCamptothecin Anticancerous Camptotheca acuminata(+)-Catechin Haemostatic Potentilla fragarioides

Drug/Chemical Action/Clinical Use Plant SourceChymopapain Proteolytic, mucolytic Carica papayaCissampeline Skeletal muscle relaxant Cissampelos pareiraCocaine Local anaesthetic Erythroxylum cocaCodeine Analgesic, antitussive Papaver somniferumColchiceine amide Antitumor agent Colchicum autumnaleColchicine Antitumor agent, anti-gout Colchicum autumnaleConvallatoxin Cardiotonic Convallaria majalisCurcumin Choleretic Curcuma longaCynarin Choleretic Cynara scolymusDanthron Laxative Cassia speciesDemecolcine Antitumor agent Colchicum autumnaleDeserpidine Antihypertensive,

tranquillizerRauvolfia canescens

Deslanoside Cardiotonic Digitalis lanataL-Dopa Anti-parkinsonism Mucuna spDigitalin Cardiotonic Digitalis purpureaDigitoxin Cardiotonic Digitalis purpureaDigoxin Cardiotonic Digitalis purpurea

Emetine Amoebicide, emetic Cephaelis ipecacuanhaEphedrine Sympathomimetic,

antihistamineEphedra sinica

Etoposide Antitumor agent Podophyllum peltatumGalanthamine Cholinesterase inhibitor Lycoris squamigeraGitalin Cardiotonic Digitalis purpureaGlaucarubin Amoebicide Simarouba glaucaGlaucine Antitussive Glaucium flavumGlasiovine Antidepressant Octea glazioviiGlycyrrhizin Sweetener, Addison's disease Glycyrrhiza glabraGossypol Male contraceptive Gossypium species

Drug/Chemical Action/Clinical Use Plant SourceHemsleyadin Bacillary dysentery Hemsleya amabilisHesperidin Capillary fragility Citrus speciesHydrastine Hemostatic, astringent Hydrastis CanadensisHyoscyamine Anticholinergic Hyoscyamus nigerIrinotecan Anticancer, antitumor agent Camptotheca acuminataKaibic acud Ascaricide Digenea simplexKawain Tranquillizer Piper methysticumKheltin Bronchodilator Ammi visageLanatosides A, B, C Cardiotonic Digitalis lanataLapachol Anticancer, antitumor Tabebuia sp.a-Lobeline Smoking deterrant,

respiratory stimulantLobelia inflate

Menthol Rubefacient Mentha speciesMethyl salicylate Rubefacient Gaultheria procumbensMonocrotaline Antitumor agent (topical) Crotalaria sessilifloraMorphine Analgesic Papaver somniferumNeoandrographolide Dysentery Andrographis paniculataNicotine Insecticide Nicotiana tabacumNordihydroguaiaretic acid Antioxidant Larrea divaricataNoscapine Antitussive Papaver somniferumOuabain Cardiotonic Strophanthus gratusPachycarpine Oxytocic Sophora pschycarpaPalmatine Antipyretic, detoxicant Coptis japonicaPapain Proteolytic, mucolytic Carica papayaPapavarine Smooth muscle relaxant Papaver somniferumPhyllodulcin Sweetner Hydrangea macrophyllaPhysostigmine Cholinesterase Inhibitor Physostigma venenosumPicrotoxin Analeptic Anamirta cocculus

Drug/Chemical Action/Clinical Use Plant SourcePilocarpine Parasympathomimetic Pilocarpus jaborandiPinitol Expectorant Several plantsPodophyllotoxin Antitumor anticancer agent Podophyllum peltatumProtoveratrines A, B Antihypertensives Veratrum albumPseudoephredrine* Sympathomimetic Ephedra sinica

Pseudoephedrine, nor- Sympathomimetic Ephedra sinicaQuinidine Antiarrhythmic Cinchona ledgerianaQuinine Antimalarial, antipyretic Cinchona ledgerianaQulsqualic acid Anthelmintic Quisqualis indicaRescinnamine Antihypertensive,

tranquillizerRauvolfia serpentine

Reserpine Antihypertensive, tranquillizer

Rauvolfia serpentine

Rhomitoxin Antihypertensive, tranquillizer

Rhododendron molle

Rorifone Antitussive Rorippa indicaRotenone Piscicide, Insecticide Lonchocarpus nicouRotundine Analagesic, sedative,

traquillizerStephania sinica

Rutin Capillary fragility Citrus speciesSalicin Analgesic Salix albaSanguinarine Dental plaque inhibitor Sanguinaria CanadensisSantonin Ascaricide Artemisia maritmaScillarin A Cardiotonic Urginea maritimeScopolamine Sedative Datura speciesSennosides A, B Laxative Cassia speciesSilymarin Antihepatotoxic Silybum marianumSparteine Oxytocic Cytisus scoparius

Drug/Chemical Action/Clinical Use Plant SourceStevioside Sweetner Stevia rebaudianaStrychnine CNS stimulant Strychnos nux-vomicaTaxol Antitumor agent Taxus brevifoliaTeniposide Antitumor agent Podophyllum peltatuma-Tetrahydrocannabinol (THC)

Antiemetic, decrease occular tension

Cannabis sativa

Tetrahydropalmatine Analgesic, sedative, traquillizer

Corydalis ambigua

Tetrandrine Antihypertensive Stephania tetrandraTheobromine Diuretic, vasodilator Theobroma cacaoTheophylline Diuretic, bronchodilator Theobroma cacao and

othersThymol Antifungal (topical) Thymus vulgarisTopotecan Antitumor, anticancer agent Camptotheca acuminata

Trichosanthin Abortifacient Trichosanthes kirilowiiTubocurarine Skeletal muscle relaxant Chondodendron

tomentosumValapotriates Sedative Valeriana officinalisVasicine Cerebral stimulant Vinca minorVinblastine Antitumor, Antileukemic

agentCatharanthus roseus

Vincristine Antitumor, Antileukemic agent

Catharanthus roseus

Yohimbine Aphrodisiac Pausinystalia yohimbeYuanhuacine Abortifacient Daphne genkwaYuanhuadine Abortifacient Daphne genkwa

1.3 Role of Plant on Cancer and Aids Research:

The National Cancer Institute (NCI) has several ongoing collaborative programs which screen plants for the possibility of new drugs and active plant chemicals for cancer and AIDS/HIV.

As well over 50 percent of the estimated 250,000 plant species found on earth come from tropical forests, NCI concentrates on these regions. Plants have been collected from the African countries of Cameroon, the Central African Republic, Gabon, Ghana, Madagascar, and Tanzania. Collections are now concentrated in Madagascar (one of the most rapidly disappearing rainforest regions in the world), and collaborative programs have been established in South Africa and Zimbabwe.

In Central and South America, samples have been collected from Belize, Bolivia, Colombia, the Dominican Republic, Ecuador, Guatemala, Guyana, Honduras, Martinique, Paraguay, Peru, and Puerto Rico. The NCI has established collaborative programs in Brazil, Costa Rica, Mexico, and Panama. Southeast Asian collections have been performed in Bangladesh, Indonesia, Laos, Malaysia, Nepal, Pakistan, Papua New Guinea, the Philippines, Taiwan, Thailand, and Vietnam. Collaborative programs have been established in Bangladesh, China, Korea, and Pakistan. In each country, NCI contractors work in close collaboration with local botanical institutions.14

Thus far seven plant-derived anticancer drugs have received Food and Drug Administration (FDA) approval for commercial production: 15

• Taxol/paclitaxel

A chemical discovered in the Pacific Yew tree (Taxus brevifolia) is now the first drug of choice in several tumorous cancers including Breast Cancer.

• Vinblastine

A chemical discovered in the Madagascar Periwinkle in the 1950s. Vinblastine is the first drug of choice in many forms of leukemia and since the 1950's it has increased the survival rate of childhood leukemias by 80%.

• Vincristine

Another antileukemic drug discovered in the Madagascar periwinkle.

• Topotecan

It has been approved by the FDA for the treatment of ovarian and small cell lung cancers. It is currently in clinical trials, either alone or in combination with other anticancer drugs, for several types of cancer. Topotecan is an analog (a synthesized chemical) of a plant alkaloid discovered in the Chinese tree species, Camptotheca acuminata.

• Irinotecan

Another chemical analog which has been developed from yet another plant alkaloid discovered in the same tree Camptotheca acuminata. It has been approved by the FDA for the treatment of metastatic colorectal cancer. It is currently in clinical trials for a variety of other cancers.

• Etoposide

It is a semi synthetic derivative of a plant chemical epipodophyllotoxin discovered in the May apple plant family (Podophyllum peltatum).

• Teniposide

It is another semi synthetic derivative of a plant chemical discovered in the May apple plant family (Podophyllum peltatum).

Since 1986, over 40,000 plant samples have been screened, but thus far only five chemicals showing significant activity against AIDS have been isolated. Three are currently in preclinical development. Before being considered for clinical trials in humans, these agents must show tolerable levels of toxicity in several animal models. For AIDS, three agents are presently in preclinical or early clinical development. The following are plants and chemicals which are still under research for cancer and AIDS/HIV:

• (+)-Calanolide A and (-)-Calanolide B (costatolide) are isolated from Calophyllum lanigerum and Calophyllum teysmanii, respectively, trees found in Sarawak, Malaysia. Both these agents are licensed to Medichem, Inc., Chicago, which is developing them in collaboration with the Sarawak State Government through a joint company, Sarawak Medichem Pharmaceuticals, Inc.(+)-Calanolide A is currently in early clinical trials in the United States.

• Conocurovone, isolated from the shrub species, Conospermum incurvum (saltbush), found in Western Australia, has been licensed for development to AMRAD, a company based in Victoria, Australia.

• Michellamine B, from the leaves of Ancistrocladus korupensis, a vine found in the Korup rainforest region of southwest Cameroon, has undergone extensive preclinical study, but is considered too toxic for advancement to clinical trials.

• Prostratin, isolated from the wood of Homolanthus nutans, a tree found in Western Samoa, has been placed on low priority, largely due to its association with a class of compounds shown to be tumor promoters.

• A tree native to China, Camptotheca acuminate, is the source of four promising anticancer drugs, two of which have been approved by the FDA and are described above. The other two chemicals still under research include:

• 9AC (9-aminocamptothecin): Currently in clinical trials for several types of cancer, including ovarian and stomach cancers and T-cell lymphoma.

• Camptothecin: While no clinical trials are being performed in the United States, trials are ongoing in China.

• Homoharringtonine from the Chinese tree, Cephalotaxus harringtonia are in early clinical trials.

• Perillyl alcohol, and flavopiridol, a totally synthetic compound based on a flavone isolated from Dysoxylum binectiferum are in early clinical trials.16-17

1.4 Bioactivity guided phytochemical investigation of the plants:

The use of plant products is increasing in many segment of the population. At present thousands of plant metabolics are being successively used in the treatment of variety of diseases. According to estimation 40% of the world’s populations rely upon plants for their medication. The use of medicinal plants is increasing in many developed countries where 35% of drugs involve natural products. Since Bangladesh has a vast resource of medicinal plants, the present study might be a significant way of making the best use of these natural resources. Majority of our population, who are impoverished, have to rely upon indigenous system of medication because of their inability to meet the cost of modern medicine. Thus in order to strengthen the existing health care system, biological activity directed chemical analysis of indigenous plant Ocimum sanctum is the primary objective of bioactivity directed phytochemical investigation of plant products. Moreover the standardization of herbal medicines has made them popular in many developed countries. So the economic impact of the present study may be reflected through export standard, high quality herbal drugs, which would increase our natural reserve. Thus the rationality of the present study lies in meeting the challenge of developing herbal medicine for the coming twenty first century which needs a systematic research on indigenous medicinal plants for the welfare of the humanity.

1.5 Objective:

Main objectives of the proposed research program are:

1. To extract and isolate biologically active compounds from the leaves of O. sanctum using various solvents such as methanol, chloroform, ethyl acetate, n-hexane and butanol and to identify the structure of the isolated compounds using various analytical and instrumental techniques including UV, IR, 1H-NMR, 13C-NMR and mass spectroscopy.

2. To evaluate the medicinal value of the Isolated Compounds obtained from the extracts of the leaves of O. sanctum by means of several studies related to antibacterial, antifungal.

3. To identify and suggest the possible area of the utilization of the isolated compounds from the extract of seeds in the cure of various diseases.

1.6 Literature Survey:

1.7 General Information:

Kingdom : PlantaeSubkingdom : Tracheobionta

Superdivision : Spermatophyta Division : Magnoliophyta

Class : MagnoliopsidaOrder : Lamiales

Family : LamiaceaeGenus : Ocimum

Species : O. sanctum

Indian Name: Tulsi Botanical or Latin Name : Ocimum sanctum English name : Sacred Basil / Holy Basil

1.8 Origin of Ocimum sanctum:Holy basil is native to tropical Asia but has been dispersed by humans so that it now grows in many tropical parts of the world. It is a sacred plant in Hindu religion, and has been cultivated in India in courtyards or temples, and in pots in homes, for about 3000 years.

Holy basil has been cultivated in India for thousands of years.

1.9 History:

The history of the plant in South Asia is closely linked with folklore and mythology. It represents Vishnupriya or Beloved of Vishnu, since it is believed to be the embodiment of the goddess Lakshmi, the spouse of Vishnu. What is apparent is that it has been valued and cultivated since ancient times in India as an intimate link between the household and the spiritual world.

The Aryans, who structured the forms of Hinduism, were nature-worshippers and their poetry and imagery were rich with the evocation of nature. Perhaps they were drawn to holy basil because of its fragrance and delicacy. It may also have been already well-entrenched in the myths of the indigenous people and from there absorbed into Hinduism.

Holy basil is mentioned in the Rig Veda, written in about 1500 BC, and its holiness is celebrated in the Puranas. It is highly regarded in the Ayurvedic system of medicine and is noted in medical treatises such the Charaka Samhita written between the 2nd century BC to the 2nd century AD.

1.10 Description of OCIMUM SANCTUM:

Tulsi plant, is a shrub reaching a height of 0.5 to 1.5 m. The leaves are 2-4 cms in length. There are several varieties of the plant. However, commonly used one is with dark leaves. The inflorescence is a long spike with tiny purple flowers. There are two varieties: a red- and a green one. Red holy basil has a stronger smell. It is cultivated in gardens throughout Bengal, /East Nepal and Deccan Peninsula; it is said to be a common wild plant in Western India.

1.11 Characteristics of Constituents:

The leaves contain an essential oil which has been studied with gas chromatography. The oil contains eugenol, eugenal, carvacrol, methylchavicol, limatrol and caryophylline. The seeds contain an oil composed of fatty acids and sitosterol. The mucilage is compared of sugars – xylose and polysaccharides.

Tulasi as an Ayurvedic medicine

Tulasi, as used in Ayurveda.

Tulasi’s extracts are used in ayurvedic remedies for common colds, headaches, stomach disorders, inflammation, heart disease, various forms of poisoning, and malaria. Traditionally, tulasi is taken in many forms: as an herbal tea, dried powder, fresh leaf, or mixed with ghee. Essential oil extracted from Karpoora Tulsi is mostly used for medicinal purposes and in herbal toiletry. For centuries, the dried leaves of Tulasi have been mixed with stored grains to repel insects.

Recent studies suggest that Tulasi may be a COX-2 inhibitor, like many modern painkillers, due to its significant amount of Eugenol (1-hydroxy-2-methoxy-4-allylbenzene).[5][6] Studies have also shown Tulsi to be effective for diabetes, by reducing blood glucose levels. [7] The same study showed significant reduction in total cholesterol levels with Tulsi. Another study showed that Tulsi's beneficial effect on blood glucose levels is due to its antioxidant properties.[8]

Tulasi also shows some promise for protection from radiation poisoning[9] and cataracts.[10]

Some Vaishnavites do not use Tulasi for medicine, though, out of reverence. However, the use of Tulsi for purification and as a medicine is widespread throughout India. Many Hindus — along with the ancient tradition of Ayurveda — believe that the healing properties of sacred herbs such as Tulsi were given by the Lord himself, and can be used as a medicine out of reverence.

1.12 Parts used and where grown

Holy basil is native to the Indian subcontinent and other parts of tropical Asia. The leaf and seed oil are used therapeutically.

Holy basil has been used in connection with the following conditions (refer to the individual health concern for complete information):

Science Ratings Health Concerns

Asthma

Type 2 diabetes

Poison oak and poison ivy dermatitis

Reliable and relatively consistent scientific data showing a substantial health benefit.Contradictory, insufficient, or preliminary studies suggesting a health benefit or

minimal health benefit.For a herb, supported by traditional use but minimal or no scientific evidence. For a

supplement, little scientific support and/or minimal health benefit.

1.13 Varieties of Holy Basil-Tulsi:

Krishna Tulsi Rama Tulsi Vana Tulsi

1.14 Active compounds:The essential oil from some populations of holy basil contains high levels of eugenol. This compound has anti-inflammatory activity, can kill bacteria and deters insects. The presence of this compound in the plant could explain why it could be used to treat pain, kill germs and provide people with some protection from being bitten by insects.

Another compound called rosmarinic acid has anti-inflammatory and antioxidant activity and these activities could contribute too many of the medicinal properties of holy basil. The plant also contains ursolic acid a compound that has been shown to provide some protection to enzymes in the liver that deal with the breakdown of fat in our diet. This is important as patients with diabetes often have high levels of cholesterol in their blood. The levels have been reported to decrease after taking holy basil.

1.15 Medicinal values of the Tulsi:

The tulsi or holy basil is an important symbol in the Hindu religious tradition and is worshipped in the morning and evening by Hindus at large. The holy basil is also a herbal remedy for a lot of common ailments. Here're top fifteen medicinal uses of tulsi.

1. Healing Power: The tulsi plant has many medicinal properties. The leaves are a nerve tonic and also sharpen memory. They promote the removal of the catarrhal matter and phlegm from the bronchial tube. The leaves strengthen the stomach and induce copious perspiration. The seed of the plant are mucilaginous.

2. Fever & Common Cold: The leaves of basil are specific for many fevers. During the rainy season, when malaria and dengue fever are widely prevalent, tender leaves, boiled with tea, act as preventive against theses diseases.

In case of acute fevers, a decoction of the leaves boiled with powdered cardamom in half a liter of water and mixed with sugar and milk brings down the temperature. The juice of tulsi leaves can be used to bring down fever. Extract of tulsi leaves in fresh water should be given every 2 to 3 hours. In between one can keep giving sips of cold water. In children, it is every effective in bringing down the temperature.

3. Coughs: Tulsi is an important constituent of many Ayurvedic cough syrups and expectorants. It helps to mobilize mucus in bronchitis and asthma. Chewing tulsi leaves relieves cold and flu.

4. Sore Throat: Water boiled with basil leaves can be taken as drink in case of sore throat. This water can also be used as a gargle.

5. Respiratory Disorder: The herb is useful in the treatment of respiratory system disorder. A decoction of the leaves, with honey and ginger is an effective remedy for bronchitis, asthma, influenza, cough and cold. A decoction of the leaves, cloves and common salt also gives immediate relief in case of influenza. They should be boiled in half a liter of water till only half the water is left and add then taken.

6. Kidney Stone: Basil has strengthening effect on the kidney. In case of renal stone the juice of basil leaves and honey, if taken regularly for 6 months it will expel them via the urinary tract.

7. Heart Disorder: Basil has a beneficial effect in cardiac disease and the weakness resulting from them. It reduces the level of blood cholesterol.

8. Children's Ailments: Common pediatric problems like cough cold, fever, diarrhea and vomiting respond favorably to the juice of basil leaves. If pustules of chicken pox delay their appearance, basil leaves taken with saffron will hasten them.

9. Stress: Basil leaves are regarded as an 'adaptogen' or anti-stress agent. Recent studies have shown that the leaves afford significant protection against stress. Even healthy persons can chew 12 leaves of basil, twice a day, to prevent stress. It purifies blood and helps prevent several common elements.

10. Mouth Infections: The leaves are quit effective for the ulcer and infections in the mouth. A few leaves chewed will cure these conditions.

11. Insect Bites: The herb is a prophylactic or preventive and curative for insect stings or bites. A teaspoonful of the juice of the leaves is taken and is repeated after a few hours. Fresh juice must also be applied to the affected parts. A paste of fresh roots is also effective in case of bites of insects and leeches.

12. Skin Disorders: Applied locally, basil juice is beneficial in the treatment of ringworm and other skin diseases. It has also been tried successfully by some naturopaths in the treatment of leucoderma.

13. Teeth Disorder: The herb is useful in teeth disorders. Its leaves, dried in the sun and powdered, can be used for brushing teeth. It can also be mixed with mustered oil to make a paste and used as toothpaste. This is very good for maintaining dental health, counteracting bad breath and for massaging the gums. It is also useful in pyorrhea and other teeth disorders.

14. Headaches: Basil makes a good medicine for headache. A decoction of the leaves can be given for this disorder. Pounded leaves mixed with sandalwood paste can also be applied on the forehead for getting relief from heat, headache, and for providing coolness in general.

15. Eye Disorders: Basil juice is an effective remedy for sore eyes and night-blindness, which is generally caused by deficiency of vitamin A. Two drops of black basil juice are put into the eyes daily at bedtime.

1.16 SafetyHoly basil has a long history of safe use in India. Application to the skin can cause reactions in sensitive people.

1.17 Chemical investigation of the genus Ocimum sanctum(Literature review) :

3. EXPERIMENTAL

3.1 Investigation On Ocimum Sanctum

3.2 Collection Of Plants

The plant Ocimum Sanctum was collected from Gazipur area and was identified from the department of Botany, Dhaka University. The collected fresh plants were cleaned thoroughly. The plant materials (leaves and stems) were separated from its roots and dried under mild sunlight and then at 40 oC in an oven. Afterwards the plants were powdered in a grinding machine (~200 smeshes). The powder was used throughout the investigation.

3.3 Phytochemical Screening Of The Plants

Chemical tests were carried out on the aqueous extract and on the powdered specimens using standard procedures to identify the constituents .

3.4 Qualitative Determination

Test For TanninsThe dried powder samples (~0.5 g) was boiled in water (20 ml) in a test tube and then filtered. A few drops of ferric chloride solution (0.1%w/v) were added and brownish green or a blue-black coloration was observed.

Test For PhlobatanninsDeposition of a red precipitate (on boiling) extract of each plant sample was boiled with aqueous hydrochloric acid (1% w/v) indicated the presence of phlobatannins.

Test For SaponinThe powdered sample (~2g) was boiled in distilled water (20 ml) in a water bath and filtered. The filtrate (10 ml) was mixed with distilled water (5 ml) and shaken vigorously for a stable persistent froth. The frothing was mixed with olive oil (3 drops) and shaken vigorously, and then the formation of emulsion was observed.

Test For FlavonoidsThree methods were used to determine the presence of flavonoids in the plant sample-a) Dilute ammonia solution (5 ml) was added to a portion of the aqueous filtrate of each plant extract followed by addition of concentrated H2SO4.b) Few drops of aluminium ion (Al3+) solution (1%) were added to a portion of each filtrate. A yellow colouration each observed indicating the prsence of flavonoids.c) A portion of the powdered plant sample each case was heated with ethyl acetate (10 ml) over a steam bath for 3 min. The mixture was filtered and the filtrate (4ml) was shaken with dilute ammonia solution (1 ml). A yellow colouration was observed indicating a positive test for flavonoids.

Test For SteroidsAcetic anhydride (2 ml) was added to ethanolic extract (0.5 g) of each sample with H2SO4

acid (2 ml). The colour changed from violet to blue or green in some samples indicating the presence of steroids.

Test For Terpenoids (Salkowski Test)

Each extract (5 ml) was mixed in chloroform (2 ml), and concentrated H2SO4 acid (3 ml) was carefully added to form a layer. A reddish brown colouration at the interface was formed to show positive test for the presence of terpenoids.

Test For Cardiac Glycosides (Keller-Killani Test)Each extracted (5 ml) was treated with glacial acetic acid (2 ml) containing ferric chloride solution (1 drop). This was underlayed with concentrated H2SO4 acid (1 ml). A brown ring at the interface indicates a deoxysugar characteristic of cardinolides. A violet ring may appear bellow the brown ring, while in the acetic acid layer, greenish ring may from just gradually throughout thin layer.

Table-3.1: Qualitative analysis of the phytochemical of the medicinal plants

Plants Alkaloid Tannin Saponin Flavonoid Steroid Terpenoid Cardiac glycoside

Ocimum Sanctum + + + + + + +

3.5 Quantitative Determination Of The Chemical Constituents

Alkaloid Determination 20

Each sample (0.5 g) was taken into a conical flask (100 ml) and acetic acid (20 ml in 10%) ethanol was added. It was covered and allowed to stand for 12 hours. This was filtered and the extract was concentrated on water bath to one-quarter of the original volume. Concentrated ammonium hydroxide was added drop wise to the extract until the precipitation was complete. The whole solution was allowed to stand and the precipitate was collected and washed with dilute ammonium hydroxide solution and then filtered. The residue is the alkaloid, which was dried and weighed.

Flavonoid Determination 22

The plant sample (1 g) was extracted repeatedly with aqueous methanol (20 ml in 80%) (CH3OH) at room temperature. The whole solution was filtered through whatman filter paper No 42 (125 mm). The filtrate was later transferred into a crucible and evaporated into dryness over a water bath ansd weighed to a constant weight.

Saponin Determination 23

Each of the powder samples (5 g) were put into a conical flask and 20% aqueous ethanol (25 ml) was added. The samples were heated over a hot water bath for 4 hours with continuous stirring at about 55oC. The mixture was filtered and the residue re-extracted with 20% aqueous ethanol (50 ml). The combined extracts were reduced to 10 ml over water bath at about 90oC. The concentrated extract (was transferred into a separatory funnel and diethyl ether (5 ml) was added and shaken vigorously. The aqueous layer was recovered while the whether layer discarded. The purification process was repeated. n-butanol (15 ml) wad added and n-butanol extracts were washed twice with aqueous 5% NaCl (3 ml of) solution). The remaining solution was heated on a water bath. After evaporation the samples were dried in an oven to a constant weight; the saponin content calculated as percentage. .

Table-3.2: Amount (% on dry powder basis) of crude alkaloid, flavonoid and saponin on the medicinal plants investigated.

Plants Alkaloids (%) Flavonoids (%) Saponin (%)

Ocimum Sanctum 0.8 14.06 8.5

3.6 Extraction And Isolation Of Compounds From Ocimum Sanctum

Plant powder was taken in a few precleaned cloth thimbles. The thimbles containing the powder were placed in a Soxhlet apparatus.

The plant powder extracted separately and exhaustively in Soxhlet apparatus first with Petroleum ether (boiling point 40-60 oC and 60-80 oC) followed by hexane. All the extracts were filtered individually; the filtrate was combined together and then concentrated in a “Buchi Rotavapor” under reduced pressure. This was shown in scheme-1

EXTRACTION SHEME OF OCIMUM SANCTUM

Powder (80g)

Extraction with pet-ether (bp.40-60oC)

Petroleum ether exract Residue

Evaporated to dryness Extracted with hexane

Gummy mass Hexane extract Residue

Evaporated to dryness

Dry mass of hexane extract (18 g)

Scheme 1: Extraction scheme of Ocimum Sanctum

3.7 Investigation Of Hexane Extract

3.8 Thin Layer Chromatography (TLC) Study

TLC analysis of the hexane extract showed the presence of one spot (Petroleum ether: Dichloromethane = 90: 20) having light brown under iodine vapor. Furthermore, TLC study showed the presence of two spots on spraying with vanillin sulfuric acid followed by heating in oven for 15 minutes. Again three spots (Dichloromethane: Ethyl acetate = 80: 20) were visible on spraying vanillin sulfuric acid followed by heating in an oven for 15 minutes. Among these three spots, two were pink and one was violet. The presence of the pink colored spot was thought to be due to the presence of steroidal or fatty acid material or both of them. It gave also positive test for steroid23.

3.9 Fractionated By Vaccum Liquid Chromatography (VLC)

The hexane extract was concentrated to dry mass about 18g by a ‘Buchi Rotavapor’. The concentrated extract was mixed with TLC grade silica gel (60 GF254). The mixture was made as powder for using in column. Then the sample was placed on the top of the bed of silica gel packed column (VLC). The column was then eluted with petroleum ether followed by mixtures of pet-ether and dichloromethane of increasing polarity. The elutes were collected in test tube with an about of about 20mL in each. Solvent system used as mobile phase in the analysis of hexane extract was listed in Table.

Table- Number of fractions collected in test tubes from VLC of hexane extract by using different solvent system

Solvent system Amounts

of solvents

(mL)

Number of test tubesPet- ether

(%)DCM

(%)

Ethyl acetate(%)

Methanol%

100 0 0 0 300 1-12

98 2 0 0 100 13-16

96 4 0 0 100 17-19

92 8 0 0 100 20-23

88 12 0 0 100 24-26

84 16 0 0 100 27-31

80 20 0 0 100 32-35

75 25 0 0 100 36-39

70 30 0 0 100 40-43

65 35 0 0 100 44-47

60 40 0 0 100 48-52

55 45 0 0 100 53-55

50 50 0 0 100 56-58

45 55 0 0 100 59-61

40 60 0 0 100 62-64

35 65 0 0 100 65-67

30 70 0 0 400 68-82

25 75 0 0 300 83-91

20 80 0 0 300 92-100

15 85 0 0 100 101-103

10 80 0 0 100 104-106

5 95 0 0 200 107-112

2 98 0 0 200 113-1180 100 0 0 100 119-121

3.10 Screening Of The Fractions

TLC monitored each of the fractions and the fractions of similar behaviors were combined together and marked as F1, F2, F3, F4, F5, F6, F7, . This is given in Table

Table-: Screening of the fractions by the similar TLC pattern

No of test tubes

Fractions Solvent system for TLC

TLC pattern

1-12 F1 90:10 (PE:DCM) One spot 17-19 F2 90:10 (PE:DCM) One spot with tailing32-35 F3 70:30 (PE:DCM) Two spots with tailing62-64 F4 20:80 (DCM:PE) One spot92-100 F5 20:80 (PE:DCM) One spot with tailing 101-103 F6 20:80 (PE:DCM) no spots 119-121 F7 20:80 (EA:DCM) two spots with tailing

3.11 Attempt Of Purification And Characterization of the Fractions

Analysis Of Fraction F1

The fraction F1 was light brown in colour. This was left undisturbed at room temperature for several days. It gave no crystals and gave an oily appearance. Then, the TLC study of the sample showed one spots on TLC plates on spraying with vanillin sulfuric acid followed by heating in an oven for 10 minutes. It was not studied further because no signal in Infra-Red Spectrum was observed.

Analysis of Fraction F2

The fraction F2 was brown in colour. The fraction was left undisturbed at room temperature for several days. . The TLC study of this showed one spot of yellow colour on spraying with vanillin sulfuric acid followed by heating in oven at 110oC for 10 minutes.

Analysis of Fraction F3

The fraction F3 left undisturbed at room temperature for several days but no crystal was obtained. This was concentrated to a deep colored semisolid. Its TLC study showed two violet spots with tailing by developing vanillin sulfuric acid . Analysis Of Fraction F4

The fraction F4 was concentrated to a small volume and was yellowish gummy material at room temperature. After sever days an gummy substance was obtained. Its TLC study showed a single spot in solvent system (Petroleum ether: Dichloromethane = 40:60). The spot was yellow and turned into violet colour by spraying with vanillin sulfuric acid.

Analysis Of Fraction F5

The fraction F5 was concentrated to a small volume and was left undisturbed at room temperature. After several days a semisolid substance was obtained. It was soluble in hexane. It’s TLC study did not give any information result.

Analysis Of Fraction Of F6

The fraction F6 was treated with charcoal to absorb chlorophyll. After treating, the fraction was filtered and left undisturbed at room temperature for several days. But no crystal was found.

Analysis Of Fraction F7

The fraction F7was treated with charcoal to absorb chlorophyll. After treating, the fraction was filtered and left undisturbed at room temperature for several days. But no crystal was found.

3.12 Properties Of The Isolated Compound

Properties of SP1

Physical state: Yellowish gummy material.

Solubility: Soluble in petroleum ether, ethyl acetate, chloroform & Di chloro methane

Amount: 10 mg.

Spectral characteristics:IR νmax (Figure- 1) cm-1(in KBr pellet)The compound SP-1 had important frequencies of absorption at 3400,2926 ,2838,2712,1720,1600,1643,1380,1254,1161,1139,1112,1041,1013,975,823,794,728,580, cm-1.

1H-NMR spectrum (CDCl3 , )

The compound SP-1 had important chemical shift at 0.85 (s, 3H, H-18), 87(s, 3H, H-26), 0.88, (s, 3H, H-27), 0.97 (t, 3H, J=7.1 Hz, H-29), 1.0 (d, 3H, J=6.5 Hz, H-21), 1.24 (s, 3H, H-

19), 2.03-3.31 (m, 3H, proton of sugar moiety), 3.86 (m, 1H, H-3), 4.85 (s, 1H, anomeric H), 4.96 (s, 1H, proton of sugar moiety), 5.03-5.08 (dd, 1H, J=12.5 and 8.5 Hz, H-22), 5.35 (dd, 1H, J-12.5 and 8.5 Hz, H-23), 5.47 (s, 1H, H-6); 6.67-6.84 (m, 1H, proton of sugar moiety), 7.25 (s, 1H, -OH);

13C-NMR spectrum ( CDCl3)The compound SP-1 had important chemical shift at 39.9 (C-1), 29.9 (C-2), 77.3 (C-3), 39.8 (C-4), 55.8 (C-5), 21.6 (C-6), 39.2 (C-7) 29.7 (C-8), 48-7 (C-9), 29.4 (C-10), 21.6 (C-11), 27.2 (C-12), 50.8 (C-13), 30.2 (C-14), 62.1 (C-15)s, 77.3 (C-16), 124.3 (C-17), 128.26 (C-18), 130.2 (C-19), 151.87 (C-20), 178.91 (C-21), 146.47 (C-22), 27.2 (C23), 14.1 (C-24), 62.12 (C-25), 76.7 (C-26), 63.75 (C-27), 184.9 (C-28), 111.14 (C-29), 121.2 (C-30), 210.3, 209.45, 130.24, 130.0, 143.96 (carbon of sugar moiety).

4. Result and discusionThe spcies ocimum sanctum of the family labiatae has been collected locally, dried, powdered and extracted with petroleum ether followed by hexane. Hexane extract of this species was fractionated by VLC (vacuum liquid chromatography) & different fractions obtained were separated & purified using different chromatographic techniques. The compounds isolated from this extract were characterized by different physical and spectroscopic studies.

4.1 Characterisation Of Compounds Isolated From Hexane Extract Of Ocimum Sanctum

4.2 Characterisation Of Compound S.P-1

The compounds S.P-1 (10 mg) was isolated as yellowish gummy material from the hexane extract of the plant Ocimum sanctum. It was soluble in hexane. TLC examination of S.P-1 indicated that it was a single compound (Rf value = 0.32 (over silica gel, GF254, petroleum ether: dichloromethane = 40:60 as the mobile phase). It was visualized as a yellow coloured spot upon its exposure to iodine vapour and as a violet coloured spot on sparaying with vanillin-sulfuric acid followed by heating in an oven at 110oC.

IR SPECTROSCOPIC STUDY

The IR spectrum of the compound S.P-1 (Figure) showed an absorption band at (3600-3400) cm-1 indicating the presence of –OH group. The band at 2900 cm-1 & 2850cm-1 were due to the presence of aliphatic C-H stretching of either both –CH3, -CH2- or >CH- group24. The peak at 1640 cm-1 was suggestive the presence of >C=C< group. The peak at 1450 cm -1 was due to the presence of –CH2- group. The absorption band at 1720 cm-1 was suggestive of C=O stretching for normal aliphatic ester. The peak at 1240 cm-1 indicated about C-N stretching. The peak at 900 cm-1 for aromatic stretching (out of plane bend). And the peaks at 830 cm -1 & 800 cm-1 was indicated the presence of >C=C-H.

1H-NMR SPECTROSCOPYThe ‘H-NMR spectrum showed two angular methyl signals at 0.85 and 1.24 indicating the terpenoid nature of the compound. r2 It also exhibited one double bond proton as a doblet at

5.35 along with two secondary methyl signals at 0.87 (H-26) and 0.88 (H-27) indicating the isooproperyl of the skeleton.

The triplet at 0.97 ppm with the intensity of 3H coupling constant 7.1 and the doublet with the intensity of 3H at 1.0 ppm and the coupling constant 6.5 were assigned for the terminal methyl group at position 29 and secondary methyl group at position 21 respectively.

The multiplet at 2.03-3.31 the presence of five protons of sugar moiety R3,4 and 7.25 was assigned proton of the OH group of glucoside.

13C-NMR SPECTROSCOPYThe 13c NMR spectrum of isolated compound revealed the presence of 29 carbons, chemical shif tat 76.7 and 63.75 were assigned for the two separate methyl group terminal carbon number 26,27 respectively. The downfield chemical shift at 128.26, 130.2, 178.91, were assigned for the angular methyl group of carbon 18, 19 and 21 respectively. The up field signal at 29.7, 29.4 and 30.2 were assignable to the carbon 8, 10, 14 those were fused. Similarly the comparatively upfield chemical shift at position 48.7, 50.8 and 55.8 ppm respectively were assigned the carbon 9, 13 and 5 those were fused in the proposed structure.

The up field chemical shift at 39.9, 29.9, 77.3, 39.8, 21.6, 39.2, 21.6, 27.2, 62.1, 77.3, were appropriate for the cyclohexyl and cyclopentyl carbons at position 1, 2, 3, 4, 6, 7, 11, 12, 15, 16, respectively. The chemical shift at 151.87, 146.47, 27.2, 14.1, 62.12, 184.9, were assigned for the carbon number 20, 22, 23, 24, 25, 28, respectively. when constitute the side chain of 6 carbon which linked at position 17 of the cyclopentyl ring.

The chemical shift at 124.3 ppm was assigned for the carbon number 17 which was the point of linkage of the side chain to the cyclopentyl ring. The very down field chemical shift at 210.3, 209.45, 130.24, 130.0, 143.96, was assigned for the carbon of the sugar moiety.

Combining IR, 1H-NMR and 13C-NMR spectroscopic data, the compound SP-1 was identified as ß-sitosteryl-D-glucoside having the structure as given below:

1

2

3

4

5

6

7

89

10

11

12

13

14 15

16

17

18

19

20

2122

23

24

25

26

27

28

29

Gluo

ß-sitosteryl-D-glucoside

It appears from literature survey that the isolation of this compound from this plant had been reported. All these spectral data were in good agreement with the reported spectral data. The reported compound SP-1 is a long chain fatty acid with some unsaturation. Such type of unsaturated fatty acid is very common in plant, so further work was not performed. However,

the presence of fatty acid make this leaves beneficial to health. It is reported the unsaturated fatty acid helps reducing blood cholesterol.

5. FATTY ACID ANALYSIS

5.1 IntroductionThe fatty acid series was so named because some of the higher members, particularly palmitic and stearic acids occur in natural fat. Fats are glycerides of fatty acids. Since glycerol is a trihydroxy alcohol and fatty acids are monobasic, a normal glyceride is a triglyceride and on hydrolysis yields three molecules of fatty acids and molecules of glycerol.

C3H5(C18H35O2)3 + 3H2O = C3H5(OH)3 + 3C18H36O2

The great numbers of the naturally occurring fatty acid belong to a few homologus series. The general formula of the fatty acids is CnH2nO2 may represent the series to which stearic acid belongs. As, however, their functional group is the carboxyl group, COOH, they are more conveniently expressed as CnH2nCOOH, since these show the nature of the funtional group. Nearly all the naturally occurring fatty acids have even number of carbon atoms in the molecule. The members of greatest importance are shown in Table

Table- : SOME COMMON FATTY ACIDS FOUND IN PLANT AND ANIMAL KINGDOM.

Common name Carbonatoms

Doublebonds

IUPAC name Source

Butyric acid 4 0 Butanoic acid Butter fatCaproic acid 6 0 Hexanoic acid Butter fatCaprylic acid 8 0 Octanoic acid Coconut oilCapric acid 10 0 Decanoic acid Coconut oilLauric acid 12 0 Dodecanoic acid Coconut oilMyristic acid 14 0 Tetradecanoic acid Plam Kernel oilPalmitic acid 16 0 Hexadecanoic acid Palm oilPalmitoleic acid 16 1 9-hexadecenoic acid Animal FatsStearic acid 18 0 Octadecanoic acid Animal fatsOleic acid 18 1 9-octadecenoic acid Olive oilVaccenic acid 18 1 11-octadecenoic acid Butter fatLinoleic acid 18 2 9,12-octadecadienoic acid Grape seed oilAlpha-linoleic acid (ALA)

18 3 9,12,15-octadecatrenoic acid

Flax seed

Gamma-linolenic acid (GLA)

18 3 6,9,12-octadecatrienoic acid

Borage oil

Arachidic acid 20 0 Eicosanoic acid Peanut oil, fish oilGadoleic acid 20 1 9-eicosanoic acid Fish oilArachidonic acid 20 4 5,8,11,14-eicosatetraenoic

acidLiver fats

EPA 20 5 5,8,11,14,17-eicosapentaenoic acid

Fish oil

Behenic acid 22 0 Docosanoic acid Rapeseed oilErucic acid 22 1 13-docosenoic acid Rapeseed oilDHA 22 6 4,7,10,13,16,19- Fish oil

docosahexaenoic acidLignoceric acid 24 0 Tetracosanoic acid Small amounts in

most fats

The fatty acids in plants are generally straight-chain compounds, ranging from three to eighteen carbons. Fatty acids containing an even number of carbon atoms are present in substantial amount because they are built up of two carbon atoms at a time, from acetic acid units. Fatty acids may be saturated or unsaturated; the saturated acids can also exist in both cis- and trans- forms.

Fatty acids isolable in water and are extracted from plant tissues by organic solvents of low polarity like petroleum ether or chloroform. They are generally found in ester linkage but are also found free and in loose molecular complexes with protein. Although alkaloids, terpenoids, steroids, flavones and their glycosides, phenols, and phenolic acids constitute a major portion of non-carbohydrate materials of a plant, fatty acids are always present in varying amounts in all plant materials.

These fatty materials may influence the handling of the plant tissues as well as any chemical treatment done on it. Therefore, the study of fatty acids, the major constitute of all fatty matters is important.

Fatty acids occur in plants in bound form25 as fats or lipids. Fats are the triglycerides of fatty acids of the same type or of the different types and yield fatty acids upon hydrolysis. Lipids are defined by their special solubility properties and are comprised of different kinds of compounds. These lipids comprise up to 7% 25of the dry weight in leaves in higher plants, about 1-15% in stems of green plant 17 and are important as membrane constitutes in the chloroplasts and mitochondria lipids also occur in considerable amounts in the seeds or fruits of a number of plants. Although numerous fatty acids are now known in plants the palmitic acid is the major saturated acid 25 in leaf lipids and also occur in varying quantities in some seeds oils. Stearic acid is the major saturated acid in seed fats of a number of plant families 25. Unsaturated acids (mainly C16 and C18) are widespread in both leaf and seed oils. A number of rear fatty acids 25 (e.g., erueic and sterculic acid) are found in seed oils of a few plants. Seed oils from plants such as olive, plam, coconut etc. are exploited commercially and are used as edible oils, for soap manufacture and in the pint industry. Fats are sometimes converted into the methyl esters of carboxylic acids by the reaction with methanol in presence of a catalytically reduced to straight-chain primary alcohols of even carbon number, and from these a host of compounds can be synthesized.

5.2 Gas Chromatography (GC) Chromatography26 is a physical process of separation in which the components to be separated are distributed between two phases a stationary phase having large surface area (for example a porous solid) and a mobile phase (a gas or liquid) moving in contact with the stationary phase which is generally coated on a porous solid phase. Different forms of chromatography are named according to the physical state of mobile phase employed; further subdivision is done according to the type of interaction between the solute and stationary phase. Now each type of chromatography can be subjected to three forms of developments; Elution, Frontal analysis or Displacement. In elution technique a carrier fluid (eluent) which is relatively invert towards the stationary phase, is kept flowing continuously and components of sample, though mutually resolved, are in mixture with the carrier fluid. Actually it is this technique that is generally employed in gas chromatography (GC) is fundamentally a

separation technique but it provides identification of a compound and with due calibration permits quantitative estimation as well. Today it is used by analytical chemists in every branch of science as a powerful analytical tool. It finds application in various fields such as gases and pollutants, petroleum products, oils and fats, foods and flavours, drugs, beverages, elemental organic analysis and a number of varied materials.

5.3 The Principle Advantages Of GCThe principal advantages of GC to an analyst are

i. The technique has strong separation power and even quite complex mixtures can be resolved into constituents.

ii. It is a micro method and only a few mg samples are enough for analysis; sensitivity of the method is very high.

iii. Speed of analysis is quite fast.iv. Gives good precision and accuracy.v. It involves relatively simple instrumentation; operation of a gas

chromatograph and related calculation do not require highly skilled personnel and thus the technique is very suitable for routine analysis.

vi. The cost of equipment is relatively low and life is generally long.

Thus in large number of instances GC is employed as the only method able to provide the desired results, in other cases it forms better alternative test method.

Any sample that can be vaporized (or the components could assume a vapor pressure of at least few mm of Hg) without thermal decomposition at the operating temperature could be analyzed by GC. At present due to various reasons, including difficult instrumentation and lack of high temperatures materials, the separating temperature is generally limited to about 4500C. Samples that cannot be vaporized are converted into volatile derivatives (for examples, fatty acids converted into methyl esters) and subjected to GC analysis.

5.4 Principle Of GC SeparationsWhen some gas or vapor comes in contact with an absorbent, a certain amount of it gets absorbed on the solid surface; according to the well known laws of Fraundilich (x/m = KC1/n), x is the mass of the vapour adsorvent in mass ‘m’ of (dissolved) in the bulk liquid; which follows the partition law of Henry (x/m = kC). Now both the sorption phenomena are selective and there are different k-values (distribution co-efficient), in general, for different vapor sorbent pairs; for the same sorbent, different vapors will have different K-values, i.e. varying affinities.

Gas chromatographic separation is accomplished in a tubular column made of glass, metal or Teflon. The column is filled with a sorbent as the stationary phase, absorbents are packed as such in the from of fine size graded powder whereas liquids are either coated as fine film on the column wall of first coated over an inert porous support such as fine powder and then packed into the column. A gas such as hydrogen, nitrogen, helium etc. serving as mobile phase, flows continuously through the column. It is called the carrier gas and serves to transport the sample components in the column. The sample is introduced as a sharp plug of vapour at the carrier gas entrance end of the column. Different components of the sample are absorbed on the stationary immediately there after, the portion of each component in the gas phase is swept further by the carrier gas and so a fraction of the sorbed amount also desorbs out to maintain the k-value; at the same time, out of the swept amount some portion will go into sorbent at the next point in the column, again to maintain the k-value. This goes on

successfully and continuously and as a sharp of, more or less Gaussian Distribution (peak shape). Now components having varying affinities for the sorbent (stationary phase) will held up in the latter to different degrees and consequently will move along the column with different speeds; if a column of sufficient length is chosen, these components will emerge out of the column at different intervals. This is how the separation of components is achieved in GC.

5.5 Procedure Of Fatty Acid Analysis 5.6 Analysis Of Fatty AcidsPetroleum ether extract (0.2731g) of Ocimum Sanctum was dissolved in petroleum ether (100ml) and extracted with 5% sodium bicarbonate solution (25mL X 2). The mixture was taken in a separatory funnel and shaken vigorously and allowed to stand for overnight. Two layers were obtained. The lower layer (aqueous) was separated and taken for the analysis of free fatty acid (FFA). The upper layer was separated and taken for the analysis of bound fatty acid (BFA).

5.7 Isolation Of Free Fatty Acid The lower part was acidified (pH 2.5) by 2M sulphuric acid. The mixture was then extracted with petroleum ether (b.p-40-600) (25mL X 3). The pet ether fraction was dried over anhydrous sodium sulphate, filtered and the filtrate was evaporated to dryness.

Now the saponified materials obtained from the pet-ether extract was taken in a pear shaped flask and 2 mL of borontrifluride-methanol (BF3-MeOH) complex was added and the mixture was refluxed on a boiling water bath for30 min. The mixture was then evaporated in a rotavapor to dryness and transferred in a small separatory funnel containing a little water (6mL). The mixture was shaken vigorously and then extracted with hexane. The aqueous layer was discarded. The hexane part containing the methyl esters of fatty acids was made free from water by adding anhydrous sodium sulphate. The solution was filtered and the filtrate was concentrated for the analysis of free acids by GLC (Shimadzu 9A, Column-BP-50, Detector- FID, 1700C-1 min/40C-2700C-30min).

5.8 Isolation Scheme For Fatty Acids From Ocimum Sanctum

Petroleum ether exact of Ocimum Sanctum Add 5% of NaHCO3

Mixture (shaken vigorously)

Transferred in separatory funnel Separate

Organic later Aqeous part (Unreacted fatty material)

BFA

Add 10mL of 0.05 M NaOH solution & shaken Reflux 30 minutes Na-salt of FFA + Glycerol

Evaporated by rotavapour Transferred in a separatory funnel+ Add distilled water + add Hexane & shake

Taken Hexane part Aqueous part is taken + add 2M H2SO4

& transferred in a separatory funnel in a beaker Add Na2SO4 & filter

Filtrate (Dryness & weighed which is total BFA)

Add 1 mg of Benzoic acid & 2mL BF3-MeOH complex

Reflux 20 minutes & evaporated

Transferred in separatory funnel & 6mL-distilled water

Add hexane & shaken, taken hexane part

Add Na2SO4 & filter

Concentrated & transferred into a vial

Send for GLC

2nd part

Aqueous part (Na-salt of fatty acid) 2M H2SO4 added to control pH 2.5

Pet ether is added and shak Transferred in a separatory funnel & taken Pet-ether part into a beaker Anhydrous Na2SO4 was added & filter Weighed pear shaped flask, transferred filtrate to it and dryness by rotavapour

Again weighed which is Total FFA

Add 1 mg of C6H5COOH & 2mL of BF3-MeOH complexReflux on boiling water bath, minimum 30 minutes

Dryness by rotavapour & transferred in a separatory funnel Add 6mL of distilled water, firstly shaken and add hexane

Taken hexane part in a beaker which is FFA of methyl ester

Add anhydrous Na2SO4, filter & concentrated by rotavapour Transferred in a vial Send for GLC

Scheme: 2 Fatty acid analysis

5.9 Isolation Of Bound Fatty Acid The upper part was taken in appear shaped flask and was added 10 mL of 0.5 M NaOH solution, was shaken well. The mixture was refluxed for 30 min in a boiling water bath. Then the mixture was evaporated to dryness by means of a rotavapour. A little water was added to the mixture and transferred to a seperatory funnel to settle down. The non-saponified materials were separated from the saponified portion (aqueous layer) by extraction with hexane.

The aqueous layer containing fatty acids (as salts) was acidified by adding 2M sulphuric acid and to pH 2.5. The mixture was extracted with hexane. The hexane part was taken in a conical flask and made from water free by adding anhydrous sodium sulphate and then filtered. The filtrate contained saponified materials.

Now the saponified materials obtained from the pet ether extract was taken in a pear shaped flask and 2 mL of borontrifluride-methanol (BF3-MeOH) complex was added and the mixture was refluxed on a boiling water bath for 3m min. The mixture was then evaporated in a rotavapor to dryness and transferred in a small separatory funnel containing a little water (6mL). The mixture was shaken vigorously and then extracted with hexane. The aqueous layer was discarded. The hexane part containing the methyl esters of fatty acids was made free from water by adding anhydrous sodium sulphate. The solution was filtered and the filtrate was concentrated for the analysis of bound fatty acids by GLC (Shimadzu 9A, Column-BP-50, Detector-FID, 1700C-1 min/40C-2700C-30min).

5.10 Result

Table Standard Retention Time (RT) of different methyl esters of different fatty acids from GC chromatogram.

Standard Retention Time (min)Fatty acid

3.62 Caprylic acid2.12 Capric acid9.36 Lauric acid6.21 Myristic acid12.21 Palmitoleic acid12.56 Palmitic acid15.19 Oleic acid15.55 Stearic acid18.31 Arachidic acid20.87 Behenic acid23.86 Lignoceric acid

Table Relative percentages of free fatty acids in petroleum ether extract of Ocimum Sanctum (Pat Shak)

Free fatty acids in petroleum ether extract of Ocimum Sanctum were not found here.

5.11 Discussion

Fat is important for many body processes and some fat are needed to eat in daily diet. Dietary fats are classified by their structure. Different types of fats react differently inside the body. Saturated fats (found mostly in animal products) increase blood cholesterol, which is a risk factor in coronary heart diseases. Unsaturated fats (found mostly in plant and marine foods) tend to lower blood cholesterol, The composition analysis of petroleum ether extract of Ocimum Sanctum by GC revealed that the bound fatty acid portion of this plant was rich in unsaturated fatty acids (such as). The plant Ocimum Sanctum contain small amount of fatty acids. Thus these plants are very important for edible purpose .Further analysis is necessary to know more about the content of fatty acids of this plant.

6. Summary

Ocimum Sanctum is one of the important medicinal plants of Labiatae family. Its local name is Tulsi. It grows Bangladesh and other East Asia, India, Australia, warmest parts of the United States and other area throughout the world. Different parts of the plant are used for the treatment of various diseases. Leaves, roots and seeds are being used by Ayurvedi, Yunani doctor and local Kabiraj. Tulsi is vary useful for aliments affecting stomach and related organs. Medicinal uses of tulsi healing power, fever of common cold, coughs, sore throat, headaches, teeth disorder, skin disorder,heart disorder.

RT (min) Fatty acid Relative percentage (%)12.51 Palmitic acid 78.4915.43 Stearic acid 21.5

As this plant grows everywhere in Bangladesh, this easily available Ocimum Sanctum plant may contribute to improve the health of people. The occurrence of seceondary metabolites in this plant vary from place to place depending on weather and soil condition. Also the occurrence of compounds varies from parts to parts of a plant.

In view of this importance in health sector we felt interested to work on phytochemical studies of its leaves and to provide information to the people elaborating its constituents and importance for preparation of the medicine. .

To extract and isolate biological active compounds from the leaves of O. sanctum using various solvents such as methanol, chloroform, ethyl acetate, n-hexane and butanol and to identify the structure of the isolated compounds using various analytical and instrumental techniques including NMR, IR and UV, mass spectroscopy.

To evaluate the medicinal value of the isolated compounds obtained from the extracts of the leaves of O. sanctum by means of several studies related to antibacterial, antifungal.

To identify and suggest the possible area of the utilization of the isolated compounds from the extract of seeds in the cure of various diseases.

For the present work, the leaves of Ocimum Sanctum were collected from the Gazipur. The leaves were dried, powdered and extracted successively in soxhlet apparatus with methanol. The crude extract was suspended in water and extracted successively with hexane, chioroform, ethyl acetate, and butanol. The hexane soluble fraction was further separated by column chromatography on silica gel with gradient systems of hexane-di chloro methane. About 10 gm of leave powder (Left after extraction with pet ether) was soaked in Hexane for few days and its TLC examination was done.

The petroleum ether extract was concentrated in vacuum and subjected to VLC over TLC grade silica gel. The elutes from VLC was monitored by TLC. According to TLC behavior the elutes gave fraction (1to7). All the fractions were concentrated and kept undisturbed for crystallization.Yellow gummy material was obtained from fraction F4, its TLC study showed a single spot in solvent system (petroleum ether: dichloromethane=40:60).The spot was yellow and into violet colour by spraying with vanillin sulfuric acid.

Its IR, 1H NMR and 13C NMR analysis showed that F4 is a long chain fatty acid with some unsaturation. Such type of unsaturated fatty acid is very common in plant, so further work was not performed. However, the presence of fatty acid make this leaves beneficial to health. It is reported the unsaturated fatty acid helps reducing blood cholesterol. According to the TLC data of the major compound is ß-sitosteryl-D-glucoside . Previously nobody reported the isolation of ß-sitosteryl-D-glucoside (SP-1) from this plant. So, ß-sitosteryl-D-glucoside (SP-1) author reported here in first time in this plant.The compound is SP-1 was identified as ß-sitosteryl-D-glucoside having the structure as given below:

1

2

3

4

5

6

7

89

10

11

12

13

14 15

16

17

18

19

20

2122

23

24

25

26

27

28

29

Gluo

- ß-sitosteryl-D-glucoside

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